The development of an anti-microbial immune response requires the coordination of different cell types, differentiation of pluripotent cells into specialized effectors and trafficking of cells to infected tissues. The kinetics and spatial organization of such events are poorly understood in vivo. Understanding these cellular and molecular interplays is critical to the design of vaccines and immunotherapeutic interventions. The requirements for helper T cell differentiation have been well studied in vitro. In vivo, recent work suggests some of the developmental events may be split in a spatial fashion: T cell cytokine commitment in the lymph node with cytokine secretion restricted to the infected tissue. The tissue factors controlling cellular function are not known but such studies argue for tissue modification of T cell responses. Current appraisal of in vivo effectors depends on ex-vivo assays that remove cells from signals provided by immune cells, tissue stroma and the inflammatory milieu and often requires in vitro stimulation using nonphysiological stimuli. Thus an accurate evaluation of Th immune function needs to include spatial location. This application is a hybrid of design and hypothesis driven research to uniquely track, in situ, an emerging antigen-specific Th2 response in mice after infection with the parasite Leishmania major. Specific aim 1 (design) seeks to develop a novel whole mount immunohistochemistry approach to visualize immune responses in situ. Specific aim 2 (hypothesis driven) uses this imaging system, combined with L. major-specific TCR transgenic cells and a fluorescent bicistronic IL-4 reporter mouse (for in situ detection of Th2 differentiation), to ask if the Th response is modified by parasite encounter in the infected tissue. The parent grant looks at the role of the T cell kinase, Irk, in modulating Th differentiation. We postulated that the failure to develop Th2 responses in the absence of Itk, in vivo, may be due in part to the failure of Th2 cells to expand, traffic or survive in the whole animal. Our third specific aim sort to track Itkdeficient T cells in vivo following L. major infection using standard immunohistochemistry on frozen tissue sections. Consistent with this R21 notice, since the submission of the parent grant, we have been developing more sophisticated techniques to image immune cell differentiation and trafficking in situ. We wish to further develop the imaging methods to track the developing immune response on infectious challenge.